Nitroreductase Research Articles

Development of a red-light emission hypoxia-sensitive two-photon fluorescent probe for in vivo nitroreductase imaging.

The overexpression of nitroreductase (NTR) in hypoxia has been recognized as a biomarker of highly aggressive disease, and the development of a hypoxia-sensitive two-photon (TP) bioimaging probe with both excitation and emission wavelengths in the red-light region provides favorable deep-tissue imaging with a low background fluorescence signal. Although quite a few TP hypoxia-sensitive fluorescent probes have been reported for NTR detection, their short emission wavelength (<550 nm) limits their application. Herein, we report a red light emissive TP hypoxia-sensitive turn-on probe (NRP) by employing Nile Red as a red-emitting fluorophore and p-nitrobenzene as an NTR recognition group with improved sensitivity. The NRP probe showed obvious strong red-fluorescence enhancement in the presence of NTR and high selectivity toward NTR in aqueous solution. Our in vitro experimental results illustrated that the NRP loaded tumor cells treated under hypoxia display remarkably strong fluorescence in both OP and TP microscopy at 655 nm with 45-fold enhancement, which affords deep-tissue penetration ability. The NRP probe was also successfully applied for imaging NTR in liver tissue slices and a 4T1-bearing mice model, which is important for bioimaging applications.

A Drug-Inducible Transgenic Zebrafish Model for Myelinating Glial Cell Ablation.

To study cellular and molecular mechanisms of demyelination and remyelination in vivo, we developed a transgenic zebrafish line, Tg(mbp:mCherry-NTR), in which expression of the bacterial enzyme nitroreductase (NTR) is driven under the myelin basic protein promoter (mbp) and thus is expressed in myelinating glia. When NTR-expressing larvae are treated with the prodrug metronidazole, the reaction between NTR and Mtz results in a toxic metabolite which selectively kills NTR-expressing cells. Using the Tg(mbp:mCherry-NTR) line, we can ablate two-thirds of oligodendrocytes following a 2-day MTZ treatment. Demyelination is evident seven days later, and remyelination is observed 16 days after Mtz treatment.The Tg(mbp:mCherry-NTR) model can be used to image cell behavior during, and to test how genetic manipulations or chemical compounds regulate, demyelination and remyelination. In this chapter, we describe the methods we used to characterize the oligodendrocyte loss, demyelination and remyelination in the Tg(mbp:mCherry-NTR) model.

Coumarin-based fluorescent 'AND' logic gate probes for the detection of homocysteine and a chosen biological analyte.

With this research we set out to develop a number of coumarin-based ‘AND’ logic fluorescence probes that were capable of detecting a chosen analyte in the presence of HCys. Probe JEG-CAB was constructed by attaching the ONOO− reactive unit, benzyl boronate ester, to a HCys/Cys reactive fluorescent probe, CAH. Similarly, the core unit CAH was functionalised with the nitroreductase (NTR) reactive p-nitrobenzyl unit to produce probe JEG-CAN. Both, JEG-CAB and JEG-CAN exhibited a significant fluorescence increase when exposed to either HCys and ONOO− (JEG-CAB) or HCys and NTR (JEG-CAN) thus demonstrating their effectiveness to function as AND logic gates for HCys and a chosen analyte.

Exploring the sequence, function, and evolutionary space of protein superfamilies using sequence similarity networks and phylogenetic reconstructions.

Integrative computational methods can facilitate the discovery of new protein functions and enzymatic reactions by enabling the observation and investigation of complex sequence-structure-function and evolutionary relationships within protein superfamilies. Here, we highlight the use of sequence similarity networks (SSNs) and phylogenetic reconstructions to map the functional divergence and evolutionary history of protein superfamilies. We exemplify this approach using the nitroreductase (NTR) flavoenzyme superfamily, demonstrating that SSN investigations can provide a rapid and effective means to classify groups of proteins, expose sequence similarity relationships across the global scale of a protein superfamily, and efficiently support detailed phylogenetic analyses. Integration of such approaches with systematic experimental characterization will expand our understanding of the functional diversity of enzymes, their evolution, and their associated physiological roles.

Improved Tol2-mediated enhancer trap identifies weakly expressed genes during liver and β cell development and regeneration in zebrafish

The liver and pancreas are two major digestive organs, and among the different cell types in them, hepatocytes and the insulin-producing β cells have roles in both health and diseases. Accordingly, clinicians and researchers are very interested in the mechanisms underlying the development and regeneration of liver and pancreatic β cells. Gene and enhancer traps such as the Tol2 transposon-based system are useful for identifying genes potentially involved in developmental processes in the zebrafish model. Here, we developed a strategy that combines a Tol2-mediated enhancer trap and the Cre/loxP system by using loxP-flanked reporters driven by β cell- or hepatocyte-specific promoters and the upstream activating sequence (UAS)-driving Cre. Two double-transgenic reporter lines, Tg(ins:loxP-CFPNTR-loxP-DsRed; 10×UAS:Cre, cryaa:Venus) and Tg(fabp10:loxP-CFPNTR-loxP-DsRed; 10×UAS:Cre, cryaa:Venus), were established to label pancreatic β cells and hepatocytes, respectively. These two double-transgenic lines were each crossed with the Tol2-enhancer trap founder lines to screen for and identify genes expressed in the β cell and hepatocytes during development. This trap system coupled with application of nitroreductase (NTR)/metronidazole (Mtz)-mediated cell ablation could identify genes expressed during regeneration. Of note, pilot enhancer traps captured transiently and weakly expressed genes such as rab3da and ensab with higher efficiencies than traditional enhancer trap systems. In conclusion, through permanent genetic labeling by Cre/loxP, this improved Tol2-mediated enhancer trap system provides a promising method to identify transiently or weakly expressed, but potentially important, genes during development and regeneration.

Cell-Permeable Fluorogenic Probes for Identification and Imaging Nitroreductases in Live Bacterial Cells.

Enzyme-activated fluorogenic probes, which invoke enzymatic catalysis to trigger the generation of fluorescence, provide a versatile platform for monitoring biological processes. The development of fluorogenic probes that can readily penetrate the cell envelopes of bacteria are essential to examine intracellular targets of live bacterial cells. Herein, we present the design, synthesis, properties, and biological applications of two series of fluorogenic probes based on cyanine 5 for identification of bacterial nitroreductase (NTR). The selected fluorogenic probe 3 generates a rapid 10-fold fluorescence response after being catalytically reduced by NTR to the intermediate para-aminobenzyl substituted which then underwent a rearrangement elimination reaction. Moreover, probe 3 is cell permeable for both Gram-positive and Gram-negative bacterial cell envelopes and is selective for NTR over other biological analytes, thus minimizing the background signal and enabling the real-time intracellular imaging of NTR in live bacterial cells.

A Highly Sensitive Chemiluminescent Probe for Detecting Nitroreductase and Imaging in Living Animals.

Hypoxia is a common characteristic of solid tumors, which is caused by the imbalance of oxygen supply and consumption. As the expression level of nitroreductase (NTR) increases in hypoxic solid tumors, NTR is one of the common biomarkers of hypoxia and widely used to evaluate the degree of tumor hypoxia. In this study, we designed and synthesized a highly water-soluble chemiluminescent probe, CL-NTR, for the detection of NTR activity in hypoxic tumors. It was found that the probe could be used to detect NTR with high sensitivity, and the total light photons increased tremendously with 6000-fold after the probe was treated with NTR. The chemiluminescence total light photons emission was directly proportional to the concentration of nitroreductase in the range of 3-55 ng/mL, with a detection limit of 0.947 ng/mL. Finally, the probe was successfully used to evaluate NTR activity in living mice by chemiluminescent imaging. In general, this probe has a remarkable response to NTR, which provides a promising method for the determination of NTR activity in vivo.

A novel fluorescent turn-on probe for highly selective detection of nitroreductase in tumor cells

The expression level of nitroreductase (NTR) is directly associated with cellular hypoxia states, which plays an important role in cancer invasion, metastasis, and resistance to treatment. Therefore, developing a facile and effective method for NTR detection is of great importance for improving anticancer treatment efficacies. To date, fluorescence method has received extensive attention for NTR detection due to its non-invasion, high sensitivity, and high spatial-temporal resolution features. However, most of fluorescent probes suffered from sophisticated systhesis procedure and poor biocompatibility, which would severely limit their practical applications especially for bioimaging. Herein, we repoted a novel chalcone-based fluorescence probe with simple preparation procedure and excellent biocompatibility for detection of NTR. The probe exhibits a highly selective fluorescence response towards NTR and the detection limit reaches 27 ng/mL. The specific fluorescence response can be ascribed to the NTR-catalyzed nitrobenzyl reduction mechanism, which has been verified by using fluorescence spectra, fluorescence lifetime, mass spectra, and inhibition experiments. Notably, this method successfully achieves hypoxia imaging in tumor cells, providing a useful tool for hypoxia pre-diagnosis.

Cell-Specific Chemical Delivery Using a Selective Nitroreductase-Nitroaryl Pair.

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. "Masking" the activity of small molecules using a general chemical modification and "unmasking" it only within target cells overcomes this limitation. To this end, we have developed a selective enzyme-substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2-nitro- N-methylimidazolyl (NM) masking group. To discover and optimize this NTR-NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

Near-infrared off-on fluorescence probe activated by NTR for in vivo hypoxia imaging

Selective and efficient detection and imaging of nitroreductase (NTR) overexpressed in hypoxic tissues is of great importance for better understanding its biological functions. The effective optical probes equipped for NTR detection in vivo is still lacking, so we developed an innovative near infrared (NIR) fluorescent on-off probe (Cy-NO2) composed of fluorescence reporting unit (an aminocyaine dye) and recognition unit (p-nitrobenzylcarbamate group). The response and mechanism of the NTR activated reduction of Cy-NO2 were evaluated in vitro through kinetic optical studies, mass spectra analysis and docking calculations. The results demonstrated that Cy-NO2 could rapidly recognize NTR with high selectivity and sensitivity. The efficient NTR detection ability of Cy7-NO2 was further validated by fluorescence imaging of hypoxic cells (A549, PC-12 and HUVEC cell). Furthermore, the in vivo hypoxia imaging by Cy-NO2 were evaluated on tumor-bearing mouse, cerebral ischemia (CIS) and deep vein thrombosis models. The results indicated a rapid and distinct enhancement of its NIR fluorescence highly appropriate for in vivomonitoring of NTR in all of the three animal models, which revealed aspiring clinical value of this NIR fluorescent hypoxia probe.

Revealing Unexplored Sequence-Function Space Using Sequence Similarity Networks.

The rapidly expanding number of protein sequences found in public databases can improve our understanding of how protein functions evolve. However, our current knowledge of protein function likely represents a small fraction of the diverse repertoire that exists in nature. Integrative computational methods can facilitate the discovery of new protein functions and enzymatic reactions through the observation and investigation of the complex sequence-structure-function relationships within protein superfamilies. Here, we highlight the use of sequence similarity networks (SSNs) to identify previously unexplored sequence and function space. We exemplify this approach using the nitroreductase (NTR) superfamily. We demonstrate that SSN investigations can provide a rapid and effective means to classify groups of proteins, therefore exposing experimentally unexplored sequences that may exhibit novel functionality. Integration of such approaches with systematic experimental characterization will expand our understanding of the functional diversity of enzymes and their associated physiological roles.

Mechanism of Two-/Four-Electron Reduction of Nitroaromatics by Oxygen-Insensitive Nitroreductases: The Role of a Non-Enzymatic Reduction Step.

Oxygen-insensitive NAD(P)H:nitroreductases (NR) reduce nitroaromatics (Ar-NO2) into hydroxylamines (Ar-NHOH) through nitroso (Ar-NO) intermediates. Ar-NO may be reduced both enzymatically and directly by reduced nicotinamide adenine dinucleotide or its phosphate NAD(P)H, however, it is unclear which process is predominant in catalysis of NRs. We found that E. coli NR-A (NfsA) oxidizes 2 mol of NADPH per mol of 2,4,6-trinitrotoluene (TNT) and 4 mol of NADPH per mol of tetryl. Addition of ascorbate, which reduces Ar-NO into Ar-NHOH, changes the stoichiometry NADPH/Ar-NO2 into 1:1 (TNT) and 2:1 (tetryl), and decreases the rate of NADPH oxidation. Ascorbate does not interfere with the oxidation of NADPH during reduction of quinones by NfsA. Our analysis of ascorbate inhibition patterns and both enzymatic and non-enzymatic reduction of nitrosobenzene suggests that direct reduction of Ar-NO by NADPH rather than enzymatic reduction is the predominant mechanism during nitroaromatic reduction.

A Redox‐Based Superoxide Generation System Using Quinone/Quinone Reductase

Superoxide (O2.- ) generation in biological systems is achieved through some of the most complex enzymatic systems. Of these, only xanthine/xanthine oxidase has been used for in vitro biochemical studies. However, it suffers from limitations such as a lack of suitable heterologous expression system for xanthine oxidase and the irreversible consumption and low solubility of xanthine under physiological conditions. Herein, we report a redox-based, enzyme-catalyzed system, in which autoxidation of hydroquinone to quinone via semiquinone results in superoxide generation. Quinone is reduced back to hydroquinone by using the NfsB (oxygen-insensitive nitroreductase) enzyme of Escherichia coli strain K-12 and nicotinamide adenine dinucleotide phosphate hydride (NADPH; which is regenerated by using the glucose/glucose dehydrogenase system). This new system relies on quinones that can be recycled and have superior water solubility, as well as enzymes that are heterologously expressed. By using a variety of quinones and reaction conditions, along with a comparison of real-time fluorescence, menadione has been identified as the optimal substrate for superoxide generation. The new redox-based system presents a viable alternative for studying the biochemistry of superoxide under different physiological and pathological conditions.

Endoplasmic reticulum-targeted two-photon turn-on fluorescent probe for nitroreductase in tumor cells and tissues

Hypoxia conditions could increase the activity of intracellular nitroreductase (NTR) and lead to many malignant diseases. Therefore, monitoring the activity of NTR is of great significance to study the related diseases. Organelles play crucial roles in the metabolism of living cells. In these organelles, the endoplasmic reticulum (ER) possesses single membrane structure, and it is the largest organelle in the cell. ER performs the synthesis, processing and modification of proteins and lipid, stabilizing the intracellular Ca2+ concentration and other physiological functions in living cells. Therefore, it is of great significance to develop ER-target probes in living system. Toward this goal, a new endoplasmic reticulum-targeted two-photon fluorescence turn-on NTR probe Na-NTR-ER is designed and synthesized. Probe Na-NTR-ER has been proved to display high sensitivity (36 ng/mL) and selectivity to NTR. Particularly, probe Na-NTR-ER has been successfully applied for the monitoring of NTR in ER with a high the Pearson's colocalization coefficient as 0.90 in HeLa cells and cancerous mouse tissues up to the depth of 100 μm with significant fluorescence signals.

A fluorescent turn-on probe for nitroreductase imaging in living cells and tissues under hypoxia conditions

Nitroreductase (NTR) plays momentous roles in biomedicine and the toxic degradation of organic nitrogen compounds mediated by cells. Numerous NTR is generated in tumor tissue due to the hypoxic conditions. The detection and monitoring of NIR in living cells and tissues can be utilized to the early diagnosis of tumors. In this paper, we developed a novel turn on fluorescent probe KC-NTR for nitroreductase detection. Taking advantage of the reducibility of nitroreductase, this probe can be transformed into the fluorescent compound KC-2 in the presence of NADH via the cascade cyclization reaction within 20 min. Our probe exhibited satisfactory sensitivity and good selectivity to NTR. A good linearity between the fluorescent intensity and the concentration of NTR ranged 0–10 μg/mL was observed, and the detection limit towards NTR was calculated to be 11 ng/mL. Furthermore, this probe was successfully employed in imaging living HepG2 cells and tumor tissues under hypoxia conditions with low cytotoxicity, indicating probe KC-NTR has great potential to monitor the NTR in the biological system.

Recent progress in small molecule fluorescent probes for nitroreductase

Nitroreductase (NTR) is a member of flavin-containing enzymes that exists widely in bacteria. Hypoxia, which is a characteristic of locally advanced solid tumors, resulting from an imbalance between oxygen consumption and supply, can result in NTR overexpression. Using either nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) as a source of reducing equivalents, NTR can catalyze the reduction of nitroaromatic compounds to the corresponding amines. Based on this reduction mechanism, NTR can be applied not only in the bioremediation and degradation of organic nitrogen compounds, but also in the development of NTR-targeted fluorescent probes to detect the hypoxic status of cancer cells. This review aims to provide a summary of the progress in fluorescent probes for NTR in recent years and elucidate the main fluorescent mechanisms that have been applied to design probes.

Two-photon fluorescent probe for detection of nitroreductase and hypoxia-specific microenvironment of cancer stem cell

Hypoxia plays a crucial role in cancer progression, and it has great significance for monitoring hypoxic level in biosystems. Cancer stem cells (CSCs) represent a small population of tumour cells that regard as the key to seed tumours. The survival of CSCs depend on the tumour microenvironment, which is distinct region has the hypoxic property. Therefore, the detection of the hypoxic CSC niche plays a pivotal role in the destructing the ‘soil’ of CSCs, and eliminating CSCs population. Numerous one-photon excited fluorescent probes have been developed to indicate the hypoxic status in tumours through the detection of nitroreductase (NTR) level. However, the biomedical application of one-photon fluorescent probes is limited due to the poor tissue penetration. In the present work, we reported a two-photon fluorescent probe to detect the NTR in CSCs and monitor the hypoxic microenvironment in vivo. The two-photon fluorescent molecular probe with a hypoxic specific response group can be reduced by NTR under hypoxic conditions. We used the two-photon probe to detect the hypoxia status of 3D cultured-CSCs in vitro and in vivo CSCs' microenvironment in tumour. The two-photon absorption cross section extends fluorescent excitation spectra to the near infrared region, which dramatically promotes the tissue penetration for hypoxic microenvironment detection of CSC in vivo.

Construction of an efficient two-photon fluorescent probe for imaging nitroreductase in live cells and tissues

Compared with traditional confocal microscopy, two-photon fluorescence microscopy (TPFM), which excites a two-photon (TP) fluorophore by near-infrared light, provides improved three-dimensional image resolution with increased tissue-image depth (>500μm) and an extended observation time. Therefore, the development of novel functional TP fluorophores has attracted great attention in recent years. Herein, a novel TP fluorophore CM-NH2, which have the donor-π-acceptor (D-π-A)-structure, was designed and synthesized. We further used this dye developed a new type of TP fluorescent probe CM-NO2 for detecting nitroreductase (NTR). Upon incubated with NTR for 15min, CM-NO2 displayed a ~90-fold fluorescence enhancement at 505nm and the maximal TP action cross-section value after reaction was detected and calculated to be 200 GM at 760nm. The probe exhibited excellent properties such as high sensitivity, high selectivity, low cytotoxicity, and high photostability. Moreover, the probe was utilized to image the tumor hypoxia in live HeLa cells. Finally, using the CM-NO2 to image NTR in tissues was demonstrated.

Informing Efforts to Develop Nitroreductase for Amine Production.

Nitroreductases (NRs) hold promise for converting nitroaromatics to aromatic amines. Nitroaromatic reduction rate increases with Hammett substituent constant for NRs from two different subgroups, confirming substrate identity as a key determinant of reactivity. Amine yields were low, but compounds yielding amines tend to have a large π system and electron withdrawing substituents. Therefore, we also assessed the prospects of varying the enzyme. Several different subgroups of NRs include members able to produce aromatic amines. Comparison of four NR subgroups shows that they provide contrasting substrate binding cavities with distinct constraints on substrate position relative to the flavin. The unique architecture of the NR dimer produces an enormous contact area which we propose provides the stabilization needed to offset the costs of insertion of the active sites between the monomers. Thus, we propose that the functional diversity included in the NR superfamily stems from the chemical versatility of the flavin cofactor in conjunction with a structure that permits tremendous active site variability. These complementary properties make NRs exceptionally promising enzymes for development for biocatalysis in prodrug activation and conversion of nitroaromatics to valuable aromatic amines. We provide a framework for identifying NRs and substrates with the greatest potential to advance.

A simple umbelliferone based fluorescent probe for the detection of nitroreductase

A simple nitrobenzyl-umbelliferone (NCOU1) was synthesised containing a nitroreductase (NTR) trigger moiety. The presence of NTR, resulted in the fragmentation of the parent molecule and release of the highly emissive fluorophore umbelliferone via an NTR-catalyzed reduction of the nitro group. In the presence of the NTR enzyme, NCOU1 gave rise to a 5-fold increase in fluorescence intensity at 455 nm and was selective for NTR over other reductive enzymes. These results indicate that NCOU1 can be used as a simple assay for the detection of NTR.